Biogenic amine flux mediated by cloned transporters stably expressed in cultured cell lines: amphetamine specificity for inhibition and efflux.

LLC-PK1 cells have been stably transfected with cDNAs encoding the human norepinephrine transporter (NET), rat dopamine transporter (DAT), and rat serotonin transporter. Using these cell lines, the specificity of each transporter toward agents that inhibit substrate influx and stimulate substrate efflux across the plasma membrane was examined. With 1-methyl-4-phenylpyridinium as a substrate for DAT and NET and serotonin as a substrate for the serotonin transporter, each transporter demonstrated a distinct pattern of inhibition by a panel of amphetamine derivatives and analogs, including amphetamine, methamphetamine (also known as "ecstasy"), p-chloroamphetamine, 3,4-methylenedioxymethamphetamine, methylphenidate (ritalin), and 5-methoxy-6-methyl-2-aminoindan. For each cell line expressing a single biogenic amine transporter, efflux of the accumulated substrate was stimulated by amphetamine derivatives, and this efflux was blocked by mazindol, an inhibitor of all three transporters. Of the amphetamine derivatives tested, some caused efflux at concentrations similar to those that inhibited transport. Other derivatives were much less effective at stimulating efflux than at inhibiting uptake. Methylphenidate caused little or no efflux, although it blocked uptake mediated by both NET and DAT. Other inhibitors of transport, such as cocaine, mazindol, citalopram, and nisoxetine, failed to stimulate efflux from these cells at concentrations that inhibited influx. The results suggest that potency toward individual plasma membrane biogenic amine transporters and the ability to release accumulated amine substrates are independent properties of each amphetamine derivative.